TW202102519A - Continuous production of recombinant proteins - Google Patents

Abstract

The present disclosure relates to methods and systems for the continuous production of recombinant proteins. In particular embodiments, the disclosure relates to methods and systems using capture chromatography, post-capture chromatography, virus filtration, and ultrafiltration/diafiltration for the continuous production of recombinant proteins.

Description

Continuous production of recombinant protein

The present disclosure relates to methods and systems for the continuous production of recombinant proteins. In certain embodiments, the present disclosure relates to methods and systems for continuous production of recombinant proteins using capture chromatography, post-capture chromatography, virus filtration, and ultrafiltration/diafiltration.

Continuous manufacturing is a routine production method used in industries such as petrochemical and food production. The benefits of continuous processes include steady state operation, reduced equipment size, high volumetric productivity, simplified process flow, low cycle time, and reduced capital costs. Konstantinov et al., J. Pharm. Sci. 104(3): 813-20 (2015).

Continuous manufacturing has not been widely implemented in the biopharmaceutical industry. Because the technological requirements for upstream and downstream development are very different, the experience of continuous downstream operations is limited. Therefore, for the biopharmaceutical industry, there is still a need for an integrated continuous biological production method that can operate for a long time and minimize operator interaction.

Provided herein is a continuous method for producing a recombinant protein, the method comprising: (a) using one or more capture chromatography systems to capture the recombinant protein from a substantially cell-free sample, and from the The recombinant protein is extracted in one or more capture chromatography systems to produce an extract containing the recombinant protein, wherein the single or multiple extracts are homogenized into a single mixture containing the recombinant protein; (b) ) Subjecting the single mixture to one or more post-capture chromatography systems and collecting the product output containing the recombinant protein, and (c) subjecting the product output to ultrafiltration and diafiltration to purify the recombinant Protein, wherein the method is integrated and continuous from step (a) to step (c).

This document also provides a manufacturing system for the production of recombinant proteins, the manufacturing system comprising: (a) a first unit operation, the first unit operation including a bioreactor containing a host cell that produces the recombinant protein, ( b) a second unit operation, the second unit operation including one or more capture chromatography systems, (c) a third unit operation, the third unit operation including one or more post-capture chromatography systems, and ( d) The fourth unit operation. The fourth unit operation includes an ultrafiltration system and a diafiltration system.

This document also provides a continuous method for the production of recombinant protein, the method comprising: (a) using one or more capture chromatography systems to capture the recombinant protein from a sample that is substantially free of cells, and from all The recombinant protein is extracted from the one or more capture chromatography systems to produce an extract containing the recombinant protein, wherein the extract is homogenized into a single mixture containing the recombinant protein, (b) Subjecting the homogenized single mixture to virus inactivation, (c) subjecting the homogenized single mixture from step (b) to one or more post-capture chromatography systems and collecting the product output containing the recombinant protein, ( d) subjecting the product output of the step to virus filtration, and (e) subjecting the product output from step (d) to ultrafiltration and diafiltration to purify the recombinant protein, wherein the method is from step ( a) to step (e) integrated and continuous.

Through the following detailed description and the scope of the patent application, and with reference to the accompanying drawings when appropriate, other aspects, embodiments and implementations of this document will become apparent.

The present disclosure relates to methods and systems for the continuous production of recombinant proteins. In certain embodiments, the present disclosure relates to methods and systems for the production of recombinant proteins using capture chromatography, post-capture chromatography, and ultrafiltration/diafiltration. The methods and systems described herein provide continuous and time efficient recombinant protein production.

As used in accordance with the present disclosure, unless otherwise indicated, all technical and scientific terms should be understood to have the same meaning as commonly understood by those skilled in the art. Unless the context requires otherwise, singular terms shall include pluralities, and plural terms shall include the singular.

In some embodiments, provided herein is a continuous method for the production of recombinant protein, the method comprising using one or more capture chromatography systems to capture the recombinant protein from a substantially cell-free sample, and from The recombinant protein is extracted from the one or more capture chromatography systems to produce an extract containing the recombinant protein, wherein the extract is homogenized into a single mixture containing the recombinant protein, and the The homogenized single mixture is passed through one or more post-capture chromatography systems and the product output containing the recombinant protein is collected, and the product output is subjected to ultrafiltration and diafiltration to purify the recombinant protein, wherein The method is integrated and runs continuously.

This document also provides a manufacturing system for the production of recombinant proteins, the manufacturing system comprising: a first operating unit, the first operating unit includes a bioreactor containing host cells that produce the recombinant protein, and a second operating unit , The second operation unit includes one or more capture chromatography systems, a third operation unit, the third operation unit includes one or more post capture chromatography systems, and a fourth operation unit, the fourth operation The unit includes an ultrafiltration system and a diafiltration system.

In some embodiments, the manufacturing system includes a fifth operating unit located between the first operating unit and the second operating unit, wherein the fifth operating unit includes a subsystem for performing virus inactivation. In some embodiments, the manufacturing system includes a sixth operating unit located between the third operating unit and the fourth operating unit, wherein the sixth operating unit includes a second subsystem for performing virus filtering. In some embodiments, the manufacturing system includes a seventh unit operation, the seventh unit operation includes a third sub-system, wherein the third sub-system includes an online excipient (in -line excipient).

As used herein, "substantially free of cells" means at least or about 90% free (e.g., at least or about 95%, 96%, 97%, 98%, or at least or about 99% free, or about 100%). % Does not contain samples of specified substances (such as mammalian cells).

In a specific embodiment, the substantially cell-free sample is obtained from a perfusion bioreactor containing host cells that produce the recombinant protein, or a fed-batch bioreactor containing host cells that produce the recombinant protein. It is removed from the reactor, or the clear liquid nutrient containing the host cell that produces the recombinant protein.

As used herein, "liquid medium" refers to a fluid that contains sufficient nutrients to allow cells to grow or proliferate in vitro. For example, the liquid medium may contain one or more of the following: amino acids (for example, 20 kinds of amino acids), purines (for example, hypoxanthine), pyrimidines (for example, thymidine), choline, inositol, sulfur Amine, folic acid, biotin, calcium, niacinamide, pyridoxine, riboflavin, thymidine, cyanocobalamin, pyruvate, lipoic acid, magnesium, glucose, sodium, potassium, iron, Copper, zinc and sodium bicarbonate. In some embodiments, the liquid culture medium may contain serum from a mammal. In some embodiments, the liquid medium does not contain mammalian serum or other extracts (a defined liquid medium). In some embodiments, the liquid medium may contain trace metals, mammalian growth hormone, and/or mammalian growth factor. Additional suitable liquid media are known in the art and are commercially available.

"Perfusion bioreactor" as used herein refers to a bioreactor that contains a plurality of cells in a first liquid medium, and the cultivation of cells present in the bioreactor includes periodic or continuous The first liquid culture medium is taken out, and at the same time or shortly thereafter, a second liquid culture medium of substantially the same volume is added to the bioreactor. In some embodiments, during the culture period (for example, the medium refeed rate on a daily basis), in an incremental time (for example, a period of about 24 hours, a period of time between about 1 minute and about 24 hours) , Or a time period greater than 24 hours), the volume of the first liquid medium taken out and added has an incremental change (for example, increase or decrease). . The ratio of the medium taken out and replaced every day can be changed according to the specific cells cultured, the initial seeding density, and the cell density at a specific time.

As used herein, a "fed-batch bioreactor" refers to a bioreactor containing multiple cells in which one or more feed streams are used to intermittently or continuously provide essential nutrients for cell growth and product formation.

As used herein, "clear liquid medium" refers to a culture of bacteria or yeast cells that is substantially free of (e.g., at least 80%, 85%, 90%, 92%, 94%, 96%, 98% , Or 99%) liquid culture medium containing no bacteria or yeast cells.

"Recombinant protein" as used herein refers to immunoglobulins, protein fragments, engineered proteins, blood factors, nanoantibodies, or enzymes, including antibodies or antibody fragments thereof.

"Unit operation" as used herein refers to the functional steps that can be performed in the method of manufacturing recombinant protein, or the elements of the system used in the process of manufacturing recombinant protein. For example, the operating unit may be filtration (for example, to remove contaminating bacteria, yeast viruses, or mycobacteria, and/or specific substances from fluids containing recombinant therapeutic proteins), capture, remove epitope tags, purify, maintain or store , Purification, virus inactivation, adjustment of the ion concentration and/or pH of the fluid containing the recombinant protein, and removal of unwanted salts.

The term "continuous method" or "continuous system" as used herein refers to a method or system in which fluid is continuously provided through at least part of a unit operation. If a unit operation can handle continuous flow input for a long time, it is continuous. Continuous unit operation has the smallest internal holding volume. The output can be continuous or discretized in small packets generated in a loop. If the method consists of integrated (physically connected) continuous unit operations (with zero or minimum holding volume between them), then it is continuous.

The "integrated method" as used herein refers to a method performed using structural elements that work together to achieve a specific result (for example, production of recombinant protein from a liquid medium).

As used herein, "extract" refers to the fluid discharged from the chromatography column or the chromatography membrane, which contains a detectable amount of recombinant protein.

"Chromatography medium" as used herein refers to the material packed into the chromatography column.

In some embodiments, the system disclosed herein is a closed system. A closed system includes unit operations that are designed and operated to limit exposure to the external environment. The material can be introduced into a closed system, but it must be added in a way that prevents the product from being exposed to the indoor environment.

The methods and systems disclosed herein include using one or more capture chromatography systems to capture the recombinant protein, and extract the recombinant protein from the one or more capture chromatography systems to produce a recombinant protein containing the recombinant protein. Extract. The chromatography medium contained in one or more capture chromatography systems can use capture mechanisms (for example, protein A binding capture mechanism, protein G binding capture mechanism, antibody or antibody fragment binding capture mechanism, substrate binding capture mechanism, cofactor Combine capture mechanism, tag bind capture mechanism, and/or aptamer bind capture mechanism) resin. In some embodiments, the one or more capture chromatography systems include a chromatography medium composed of resin beads, porous membranes, or nanofibers. In some embodiments, the chromatography medium is functionalized for affinity chromatography, cation exchange chromatography, anion exchange chromatography, hydrophobic interaction chromatography, or mixed mode chromatography. In some embodiments, the affinity chromatography medium is a protein A-based resin.

In some embodiments, the one or more capture chromatography systems are periodic counter current chromatography systems (PCCS). In some embodiments, the PCCS includes two chromatography columns or includes a modified ÄKTA system (GE Healthcare, Piscataway, NJ), which can be configured with up to four, five, six, seven One, eight or more than eight pipe strings to run. In some embodiments, PCCS utilizes a string switching mechanism. The column switching event can be detected by detecting the level of the recombinant protein (detected by the UV absorbance corresponding to a certain level of the recombinant protein in the fluid passing through the chromatography system), the specific volume of the liquid (for example, buffer), or Trigger after a specific time has passed.

In order to use the capture chromatography system to capture the recombinant protein, the chromatographic steps of loading, washing, extraction, and regeneration of the capture chromatography system must be performed sequentially.

In some embodiments, the extract from the one or more capture chromatography systems is homogenized into a single mixture containing the recombinant protein. In some embodiments, the homogenized single mixture is passed through one or more post-capture chromatography systems, and the product output containing the recombinant protein is collected. After use, the capture chromatography system removes remaining traces or small amounts of contaminants or impurities from the fluid containing the recombinant protein close to the final desired purity. In some embodiments, the one or more post-capture chromatography systems include a chromatography medium composed of resin beads, porous membranes, or nanofibers. In some embodiments, the chromatography medium is functionalized for affinity chromatography, cation exchange chromatography, anion exchange chromatography, hydrophobic interaction chromatography, or mixed mode chromatography. In some embodiments, the one or more post-capture chromatography columns are periodic countercurrent chromatography systems (PCCS).

In order to use the post-capture chromatography system to post-capture the recombinant protein, the chromatographic steps of loading, washing, extraction, and regeneration of the post-capture chromatography system must be carried out in sequence.

In some embodiments, the methods and systems include ultrafiltration (UF) and/or diafiltration (DF) to further purify and concentrate the recombinant protein. UF/DF can increase the concentration of recombinant protein and replace buffer salts with specific formulation buffers. Ultrafiltration (UF) refers to a type of membrane filtration in which hydrostatic pressure forces the liquid against a semi-permeable membrane. In some embodiments, UF is performed with tangential flow filtration (TFF), which includes single TFF and high performance tangential flow filtration (HPTFF). Single-pass tangential flow filtration (SPTFF) refers to any kind of TFF system in which the conversion rate (permeate flow rate divided by the inlet feed flow rate) of a single pass through the module is large enough so that the system can be used without retentate recovery (Recirculation) to operate in the case of a loop. SPTFF can be used to perform on-line concentration between other unit operations, for example to reduce the processing volume before the chromatography or precipitation step. Zydney, Biotechnol. Bioeng. 113(3): 465-75 (2016).

"Diafiltration" as used herein refers to a method of removing, replacing or reducing the concentration of salts or buffer components from a solution containing proteins such as antibodies, peptides, nucleic acids, and other biomolecules using ultrafiltration membranes. Continuous diafiltration (also called constant volume diafiltration) involves washing out the original buffer salt (or other low molecular weight species) in the retentate by adding water or a new buffer (such as a preparation buffer) to the retentate , To form a formulation containing the recombinantly produced polypeptide. Typically, the new buffer is added at the same rate as the filtrate is produced, so that during diafiltration, the retentate volume and product concentration do not change significantly. In a specific embodiment, the diafiltration is performed using a single-pass diafiltration cartridge.

In some embodiments, the homogenized single mixture is subjected to virus inactivation before being subjected to one or more post-capture chromatography systems. In some embodiments, virus inactivation includes solvent-detergent solution inactivation, heat inactivation, or acid pH inactivation.

For solvent-detergent virus inactivation, the procedure involves subjecting the sample containing the recombinant protein to an organic solvent and detergent. The solvent-detergent combination can be any solvent-detergent combination known in the art, such as tri-n-butyl phosphate and Triton X-100™, Tween 80™ and sodium cholate and others.

Virus inactivation by heat inactivation involves subjecting the sample containing the recombinant protein to a higher temperature. In some embodiments, the method includes heating the sample to a temperature greater than or equal to 45°C, 46°C, 47°C, 48°C, and at most about greater than or equal to 49°C, 50°C, 51°C, and higher. In some embodiments, the sample is heated to a temperature between 45°C and 65°C.

The heating time of the sample can vary. For example, in some embodiments, the sample is heated to the target temperature for a time between 1 minute and 6 hours. In some embodiments, the sample is heated to the target temperature for a time between 10 and 180 minutes, between 20 and 180 minutes, between 20 and 60 minutes, or between 20 and 40 minutes.

In some embodiments, the acidic pH inactivation is performed by adjusting the homogenized single mixture to a low pH with one or more solutions, and incubating the adjusted homogenized single mixture at the low pH A period of time to produce a virus inactivation mixture.

The methods and systems disclosed herein include online monitoring, including detecting the level of recombinant protein (detected by UV absorbance), detecting flow rate, and/or detecting the volume of fluid (such as buffer). Monitoring recombinant protein concentration (for example, by UV monitoring) can be determined by any tool with feedback control that can measure product concentration online.

In some embodiments, the homogenized single mixture is exposed to low pH for 15 minutes to 2 hours. In a specific embodiment, the low pH is a pH between 3 and 5. The choice of pH level depends on the stability profile of the recombinant protein and other buffer components. After the virus is inactivated, the pH of the antibody solution can be adjusted to a more neutral pH, for example between 4.0 and 8.5, before continuing the method.

In some embodiments, virus inactivation is performed in a tubular flow reactor, as described in Parker et al., Biotechnol. Bioeng. 115(3): 606-16 (2018). In some embodiments, the tubular flow reactor has a defined minimum residence time of at least 30 minutes.

In some embodiments, a virus removal step, such as virus filtration, is included after collecting the product output from the post-capture chromatography system. A virus removal step is performed to remove small non-enveloped viruses that are more resistant to virus inactivation treatment. In some embodiments, virus filtration is performed in a pressurized circuit, wherein the pressurized circuit includes a pressure vessel, a virus removal filter, and a sterilization filter. In some embodiments, the virus removal filter in the pressurized circuit is a hollow fiber virus filter. In some embodiments, the pressure vessel is a single-use, airtight and sterilizable vessel. As used herein, the term "sterilizable" refers to vessels formed from materials compatible with known sterilization methods. In some embodiments, closed-end filtration is used for virus filtration. As used herein, "closed-end filtration" refers to filtration in which the entire fluid flow to be filtered passes through the filter without recovery or retentate flow. In some embodiments, the virus removal filter is an ultra filter or a nano filter. Examples of suitable virus filtration systems are disclosed in International Publication No. WO 2018/035116, which is incorporated herein by reference.

In some embodiments, the first unit operation includes an outlet connected to an inlet on the second unit operation, wherein the second unit operation includes an outlet connected to an inlet on the third unit operation , Wherein the third unit operation includes an outlet connected to the inlet on the fourth unit operation. In some embodiments, the first unit operation includes an outlet connected to the inlet on the fifth unit operation, wherein the fifth unit operation includes an outlet connected to the inlet on the second unit operation , Wherein the second unit operation includes an outlet connected to the inlet of the third unit operation, wherein the third unit operation includes an outlet connected to the inlet of the sixth unit operation, wherein the first unit operation The six unit operation includes an outlet connected to the inlet on the fourth unit operation.

In some embodiments, the system disclosed herein includes a buffer vessel between each of the first unit operation, the second unit operation, the third unit operation, and the fourth unit operation. The buffer dish can hold any liquid culture before moving into the next operating unit.

The system described herein may also include fluid conduits arranged between any unit operations. Suitable fluid conduits can be tubes made of polyethylene, polycarbonate or plastic. The fluid conduit may also include one or more of the following in any combination: one or more online buffer adjustment reservoirs, which are in fluid communication with the fluid conduit, and are positioned such that they will be stored in the one or more online buffer adjustment reservoirs The buffer in the container is added to the fluid present in the fluid conduit; and one or more filters are arranged in the fluid conduit so that they can filter (for example, remove bacteria) the fluid present in the fluid conduit.

In some embodiments, the system provided herein includes a pump system. The pump system may include one or more of the following: one or more pumps known in the art, one or more filters known in the art, and one or more UV detectors.

In some embodiments, after ultrafiltration/diafiltration, one or more excipients are added to the product output to produce a therapeutic drug substance.

As used herein, "therapeutic drug substance" refers to a substance that contains recombinant protein that has been sufficiently purified or that has been combined with contaminating proteins, lipids, and nucleic acids (for example, contaminating proteins, lipids, and nucleic acids present in a liquid culture medium). Nucleic acids are either separated from host cells (e.g., mammalian, yeast, or bacterial host cells) and biological contaminants (e.g., viral and bacterial contaminants), and can be formulated into pharmaceuticals without any further substantial purification and/or Purification step.

In some embodiments, the system disclosed herein is on a slide rail. "Slide rail" as used herein refers to a three-dimensional solid structure that can serve as a platform or support for the system described herein. If the slide rail includes one or more movable structures (eg, wheels, rollers, etc.), it can give mobility to the system or a part of it.

In some embodiments, the methods and systems have at least about 40%, 50%, 55%, or 60%, and at most about 65%, 70%, 75%, 80%, 85%, 90%, or 95% % Recovery rate of recombinant protein. In some embodiments, the purification recovery is at least about 60% to about 70%.

In some embodiments, the methods and systems described herein result in a recombinant protein in a therapeutic protein drug substance with a net yield of at least about 5 days, 10 days, 20 days, or 30 days, and at most about at least about 280 Within a continuous period of days, 290 days, 300 days, 310 days, 320 days, 330 days, 340 days, 350 days, or 365 days, at least about 5 g/day, 10 g/day or 20 g/day, 30 g/day or 40 g/day, and up to about at least about 200 g/day, 300 g/day, 400 g/day, 500 g/day, or 1000 g/day. Instance

The following examples illustrate specific embodiments of the present disclosure and their various uses. The examples are set forth for explanatory purposes only, and should not be construed as limiting the scope of the present disclosure in any way. Example 1 : Continuous production of recombinant protein

A 100-L enhanced perfusion bioreactor coupled with dual ATFs was used to produce recombinant monoclonal antibodies (mAb) in the course of 70 days. The cell-free and mAb-containing medium (harvest) was continuously pumped to a 100 L harvest buffer dish for further processing.

The antibody-containing medium is captured using a 2X 1-L (8 x 20 cm) prepackaged and irradiated protein A affinity chromatography column, which is in the bind-and-elute MCC mode (bind-and-elute MCC mode). ) And operate on a pilot scale steam-in-place PCC slide. The capture step is performed so that one of the two columns is always loaded from the harvest buffer dish, and the average inlet volume flow rate entering the capture step matches the average outlet volume flow rate leaving the perfusion bioreactor. By Delta UV, the load on the protein A column is controlled at a breakthrough of about 3%. Before virus inactivation, the respective capture column eluate was collected in a 5-L mixing vessel. The protein A operating system was integrated with the perfusion bioreactor on the 12th day, and it was operated continuously for 58 days. Figures 3A to 3F show data summarizing the performance of protein A.

The respective protein A extracts are mixed in a 5-L mixing vessel to ensure a homogeneous flow for the virus inactivation unit operation. The outflow of the mixing vessel is automated, so that the entire vessel is processed before the next protein A extraction loop. The homogenized protein A extract was adjusted online by adding 1 M acetic acid to adjust its target pH to 3.6. Then pump the low-pH-adjusted protein A extract through a tubular flow reactor (TFR), where the minimum residence time is ≥ 30 minutes to ensure that there is enough time at low pH to achieve complete virus inactivation . Then, the virus-inactivated protein A extract was adjusted online to its target pH of 4.5 by adding 0.75 M sodium acetate. The conditioned virus-inactivated protein A extract is then processed through a sterile grade filter and continuously collected in a small buffer dish for further processing. The virus inactivation operation was integrated with the previous operation on the 15th day, and the operation was continued for 55 days. Figures 4A to 4G show data summarizing the performance of virus inactivation operations.

The adjusted virus-inactivated protein A extract was further processed through two orthogonal chromatography operations using mixed mode anion exchange resin and hydrophobic interaction chromatography resin, operating in flow-through mode. For this example process, there is no inter-step adjustment between the two post-capture chromatography operations, allowing the flow-through to be directly loaded from the mixed mode column onto the hydrophobic interaction column. The post capture train consists of 2X 0.2 L (5 x 10 cm) pre-packed mixed mode column and 2X 0.2 L (5 x 10 cm) pre-packed hydrophobic interaction column, which is operated with protein A The same, the system operates in MCC mode on the PCC slide rail. The conditioned and virus-inactivated protein A eluent is continuously loaded on one of the two parallel post-capture trains, where the column load is automated so that the load in each post-capture loop is equivalent to approximately 2 The quality of the protein A extract.

The combined mixed mode/hydrophobic interaction flow-through (labeled as post-capture extract) is continuously collected in a small buffer dish for further processing. The post-capture chromatography operation was integrated with the previous unit operation on the 23rd day, and the operation continued for 47 days. Figures 5A-5D show the summary of the captured chromatographic performance data.

The post-capture extract was passed through a 0.1 m ² Planova 20N (AK Bio) virus removal filter for further processing, which was operated in a tangential flow mode. The post-capture eluate is continuously transferred from the buffer vessel to a pressurized circuit, which contains a pressure vessel, a virus removal filter, a sterilization-grade filter, and a peristaltic pump to drive and recirculate, as shown in Figure 6A Show. The pressure in the loop is automated, so the net volume flow rate (permeate flux) through the virus filter is equal to the net volume flow rate from the post-capture operation. Based on preliminary virus verification studies, Planova 20N filters are periodically replaced. The virus-filtered material is continuously collected in small buffer dishes for further processing. The virus filtering operation was integrated with the previous unit operation on the 29th day, and the operation continued for 41 days. Figures 6B to 6F show data summarizing the performance of virus filtration operations.

The virus-filtered material was ^{further processed by ultrafiltration using a 0.065 m 2} ILC single-pass TFF (SP-TFF) box, and concentrated to a target mAb concentration of 65 g/L. The material is pumped from the post virus filter buffer dish to the inlet of the SP-TFF box, and its volumetric flow rate matches the outlet flow rate leaving the virus filtration operation. The feedback loop is used to automatically control the flow rate of the retentate leaving the SP-TFF box by measuring the product concentration online to maintain the target concentration value. The concentrated mAb stream is then directly pumped to the inlet of the single-pass diafiltration (SP-DF) cartridge for online buffer exchange. The diafiltration buffer is pumped to the buffer inlet of the SP-DF box, and the buffer flow is automated to keep the ratio of buffer to product flow constant. Control the retentate flow rate to match the inlet flow rate. Then the concentrated excipient solution is added online to prepare the buffer exchanged DF retentate stream to produce the formulated material. The formulated materials are further processed through sterile-grade filters to produce drug substances, which are collected in single-use containers. The ultrafiltration and diafiltration operations were integrated with the previous unit operations on the 42nd day, and the operation continued for 29 days. On the 46th day, the compounding operation and the drug substance production were integrated, and the operation was continued for 25 days. In the course of 25 days, 6 batches of drug substance were produced. Figures 7A to 7E and Figures 8A to 8D show data summarizing the performance of ultrafiltration and diafiltration operations. Figures 9A to 9H show the properties of the drug substance produced. Example 2 : Continuous production of recombinant protein

A 100-L enhanced perfusion bioreactor coupled with dual ATFs was used to produce recombinant monoclonal antibodies (mAb) in the course of 27 days. The cell-free and mAb-containing medium (harvest) was continuously pumped to a 100 L harvest buffer dish for further processing.

The antibody-containing medium is captured using a 2X 1-L (8 x 20 cm) prepackaged and irradiated protein A affinity chromatography column, which is operated in the binding and extraction MCC mode, and is used in the pilot scale. Steam-in-place PCC slides. The capture step is performed so that one of the two columns is always loaded from the harvest buffer dish, and the average inlet volume flow rate entering the capture step matches the average outlet volume flow rate leaving the perfusion bioreactor. By Delta UV, the load on the protein A column is controlled at about 3% breakthrough amount. Before virus inactivation, the respective capture column eluate was collected in a 5-L mixing vessel. The protein A operation was integrated with the perfusion bioreactor on the 12th day, and the operation was continued for 15 days.

The respective protein A extracts are mixed in a 5-L mixing vessel to ensure a homogeneous flow for the virus inactivation unit operation. The outflow of the mixing vessel is automated, so that the entire vessel is processed before the next protein A extraction loop. The homogenized protein A extract was adjusted online by adding 1 M acetic acid to adjust its target pH to 3.6. Then, the low-pH-adjusted protein A extract is pumped through a tubular flow reactor (TFR) printed in 3D and irradiated with γ-rays. The minimum residence time is ≥ 30 minutes to ensure low There is enough time at pH to achieve complete virus inactivation. Then, the virus-inactivated protein A extract was adjusted online to its target pH to 4.5 by adding 0.75 M sodium acetate. Then, the conditioned virus-inactivated protein A extract is processed through a sterile grade filter, and is continuously collected in a small buffer dish for further processing. The virus inactivation operation was integrated with the previous operation on the 13th day, and the operation was continued for 14 days.

The adjusted virus-inactivated protein A extract was further processed by two orthogonal chromatography operations, which used mixed mode anion exchange resin and hydrophobic interaction chromatography resin, and operated in flow-through mode. The post-capture column is composed of 2X 0.2 L (5 x 10 cm) pre-filled and γ-ray radiation irradiated mixed-mode tube columns operating in MCC mode and 2X 0.2 L (5 x 10 cm) pre-filled and γ-ray radiation irradiated It is composed of the hydrophobic interaction column. The conditioned and virus-inactivated protein A eluent is continuously loaded on one of the two mixed mode columns, and the flow-through of the mixed mode column is continuously loaded on the two hydrophobic interaction columns Enter one of them. Load the mixed mode column and the hydrophobic interaction column to their respective load capacities.

The post-capture chromatography operation was integrated with the previous unit operation on the 14th day, and the operation was continued for 13 days.

The captured eluate is passed through a 0.1 m ² Planova 20N (AK Bio) virus removal filter for further processing, which is operated in a tangential flow mode. The captured eluate is continuously transferred from the outlet of the hydrophobic interaction column to a pressurized circuit, which contains a pressure vessel, a virus removal filter, a sterilization-grade filter, and a peristaltic pump to drive and return ring. The pressure in the loop is automated, so the net volume flow rate (permeate flux) through the virus filter is equal to the net volume flow rate from the post-capture operation. The virus-filtered material is continuously collected in small buffer dishes for further processing. The virus filtering operation was integrated with the previous unit operation on the 14th day, and the operation was continued for 13 days.

The virus-filtered material was ^{further processed by ultrafiltration using tandem 2x0.1 m 2} γ-ray radiation irradiated TFF capsules, which were operated in a single pass mode. The virus filtered material was concentrated to a target mAb concentration of 110 g/L. The material is pumped from the post-virus filter buffer dish to the inlet of the TFF capsule, and its volumetric flow rate matches the outlet flow rate from the virus filtration operation. The feedback loop is used to measure the product concentration online to automatically control the flow rate of the retentate leaving the TFF capsule to maintain the target concentration value. The concentrated mAb stream is then directly pumped to the inlet of a single-pass diafiltration (SP-DF) cartridge irradiated with gamma radiation for online buffer exchange. The diafiltration buffer is pumped to the buffer inlet of the SP-DF box, and the buffer flow is automated, and the ratio of buffer to product flow is kept constant. Control the retentate flow rate to match the inlet flow rate. Then the concentrated excipient solution is added online to prepare the buffer exchanged DF retentate stream to produce the formulated material. The addition of the formulated buffer is controlled by the feedback loop and the online sensor (which measures the excipient level in the formulated stream). The formulated material is further processed through a sterile-grade filter to produce a drug substance, which is collected in a single-use container. The ultrafiltration and diafiltration operations were integrated with the previous unit operations on the 18th day, and the deployment operations were integrated on the 20th day. Due to the problem of the online sensor, the UF/DF and preparation operations did not reach a stable state before the end of the campaign.

Although this document has been described based on various embodiments, it should be understood that changes and modifications will occur to those skilled in the art. Therefore, the scope of the attached patent application is intended to cover all such equivalent changes within the scope of this document as requested. In addition, the chapter titles used in this article are for architectural purposes only and are not construed as limiting the subject described.

Unless there is a clear indication to the contrary, each embodiment described herein can be combined with any other one or more embodiments. In particular, unless there is a clear indication to the contrary, any feature or embodiment indicated as preferred or advantageous can be combined with any other feature or embodiments or one or more embodiments indicated as preferred or advantageous. .

All references cited in this application are explicitly included as part of the content disclosed in this article by citation.

no.

Figure 1 is a schematic diagram showing the process flow of the method disclosed herein. The process flow includes the continuous process of using online buffer to produce recombinant protein, which includes: (1) The production of recombinant protein in a bioreactor coupled with dual alternating tangential flow (ATF) filters and cell separation (labeled as "BRX"), (2) To capture recombinant protein, use one or more capture chromatography systems to capture the recombinant protein, the recombinant protein is present in the medium obtained in the bioreactor connected to the capture chromatography system (labeled " Capture”), (3) Virus inactivation of the medium (marked as “VI”), (4) Pass the medium through one or more post-capture chromatography systems (marked as “post-capture”), (5) Virus filtration, and (6) Ultrafiltration/diafiltration of medium.

Figures 2A and 2B show an overview of the unit operation integration of the method and system disclosed herein. Figure 2A shows the unit operation integration duration of the continuous operation of each operation unit. Figure 2B shows the average protein residence time for each unit operation. The methods and systems disclosed herein produce approximately 5 kg of drug substance over a 25-day period.

Figures 3A to 3F are graphs showing the protein A performance of the method and system disclosed herein. Figure 3A shows the concentration of protein A extract (g/L), Figure 3B shows the host cell protein remaining in the protein A extract, Figure 3C shows the residual protein in the protein A extract, and Figure 3D shows the inlet mass flow of protein A, and Figures 3E and 3F show protein A UV chromatograms.

4A to 4G are graphs showing the performance of virus inactivation operation of the method and system disclosed herein. Figure 4A shows the virus inactivation pH maintained throughout the unit operation, Figure 4B shows the protein concentration before inactivation, Figure 4C shows the protein mass flow during virus inactivation, and Figure 4D shows the acid flow during virus inactivation. Figure 4E shows the alkali flux during virus inactivation, Figure 4F shows the high molecular weight species during virus inactivation, and Figure 4G shows the protein concentration after virus inactivation.

Figures 5A to 5D are graphs showing the post-capture operating performance of the method and system disclosed herein. Figure 5A shows the post-capture eluate residual protein A, Figure 5B shows the post-capture eluate protein concentration, Figure 5C shows the UV chromatogram, and Figure 5D shows the post-capture eluate residual host cell protein.

Figure 6A shows a schematic diagram of virus filtration of the methods and systems disclosed herein. 6B to 6G are graphs showing the performance of virus filtration operation of the method and system disclosed herein. Figure 6B shows the protein concentration after virus filtration, Figure 6C shows the high molecular weight species after virus filtration, Figure 6D shows the protein mass flux during virus filtration, and Figure 6E shows the filtrate volume flux during virus filtration. 6F shows the transmembrane pressure during virus filtration, and Figure 6G shows the filter performance during virus filtration.

Figures 7A to 7E are graphs showing the ultrafiltration operating performance of the methods and systems disclosed herein. Figure 7A shows the protein concentration and conversion rate after ultrafiltration, Figure 7B shows the filter performance during the ultrafiltration, Figure 7C shows the inlet protein mass flux during the ultrafiltration, and Figure 7D shows the permeate during the ultrafiltration Volume flux, and Figure 7E shows the transmembrane pressure during ultrafiltration.

8A to 8E are graphs showing the performance of the diafiltration operation of the method and system disclosed herein. Figure 8A shows the permeate volume flux during diafiltration, Figure 8B shows the protein mass flux during diafiltration, Figure 8C shows the transmembrane pressure during diafiltration, Figure 8D shows the diafiltration ratio, and Figure 8E shows Filter performance during diafiltration.

Figures 9A to 9H are graphs showing the properties of pharmaceutical substances produced using the methods and systems disclosed herein. Figure 9A shows the concentration of the drug substance, Figure 9B shows the high molecular weight species of the drug substance, Figure 9C shows the charge characteristic curve of the drug substance, Figure 9D shows the osmolality of the drug substance, Figure 9E shows the pH of the drug substance, and Figure 9F shows the ELISA measures the host cell protein of the drug substance, Fig. 9G shows the non-reducing purity of the drug substance, and Fig. 9H shows the residual protein A of the drug substance.

Claims (41)

A continuous method for producing recombinant protein, the method comprising: (a) Use one or more capture chromatography systems to capture the recombinant protein from a sample that is substantially free of cells, and extract the recombinant protein from the one or more capture chromatography systems to produce The extract of the recombinant protein, wherein the extract is homogenized into a single mixture containing the recombinant protein; (b) Pass the homogenized single mixture through one or more post-capture chromatography systems, and collect the product output containing the recombinant protein; and (c) Subjecting the product output to ultrafiltration and diafiltration to purify the recombinant protein; The method is integrated and continuous from step (a) to step (c). The method of claim 1, further comprising subjecting the homogenized single mixture to virus inactivation after step (a). The method according to claim 2, wherein the virus inactivation includes solvent-detergent solution inactivation, heat inactivation, or acid pH inactivation. The method according to claim 3, wherein the acidic pH inactivation comprises: (a) Adjust the homogenized single mixture online to a low pH by adding one or more solutions; and (b) The adjusted homogenized single mixture is incubated at the low pH for a period of time to produce a virus-inactivated mixture. The method according to claim 4, wherein the time is between 15 minutes and 2 hours. The method according to claim 1, further comprising subjecting the product output of step (b) to virus filtration. The method according to claim 1, wherein the substantially cell-free sample is obtained from a perfusion bioreactor containing host cells that produce the recombinant protein, and a batch supplement containing host cells that produce the recombinant protein. It is removed from a feed-type bioreactor or a clear liquid culture containing host cells that produce the recombinant protein. The method according to claim 1, wherein the one or more capture chromatography systems and the one or more post-capture chromatography systems include chromatography media, and the chromatography media includes resin beads, porous membranes, or Nano fiber. The method according to claim 8, wherein the chromatography medium is functionalized for affinity chromatography, cation exchange chromatography, anion exchange chromatography, hydrophobic interaction chromatography, or mixed mode chromatography. The method according to claim 9, wherein the affinity chromatography medium is a protein A-based resin. The method according to claim 1, wherein the one or more capture chromatography systems and the one or more post-capture chromatography systems are periodic counter current chromatography systems (PCCS). The method according to claim 4, wherein culturing the adjusted homogenized single mixture at the low pH for a period of time to produce virus inactivation is performed in a tubular flow reactor. The method according to claim 4, wherein the addition of the one or more solutions is based on online recombinant protein concentration measurement and online flow rate measurement. The method according to claim 6, wherein the virus filtering is performed in a pressurized circuit. The method of claim 14, wherein the pressurization circuit includes a pressure vessel, a virus removal filter, and a sterilization filter. The method according to claim 15, wherein the pressure vessel is a single-use, airtight and sterilizable vessel. The method according to claim 16, wherein the virus removal filter is an ultra filter or a nano filter. The method according to claim 6, wherein the virus filtering is performed using closed-end filtering. The method according to claim 18, wherein the closed-end filtering is performed using an ultra filter or a nano filter. The method according to claim 1, wherein the ultrafiltration is performed by single-pass tangential flow filtration. The method according to claim 1, wherein the diafiltration is performed using a single-pass diafiltration cartridge. The method according to claim 1, wherein the recombinant protein is an antibody or an antigen-binding fragment thereof. The method according to claim 1, which further comprises adding one or more excipients after step (c) to produce a therapeutic drug substance. A manufacturing system for the production of recombinant proteins, the manufacturing system comprising: (a) The first unit operation, where the first unit operation includes a bioreactor containing host cells that produce the recombinant protein; (b) The second unit operation, where the second unit operation includes one or more capture chromatography systems; (c) The third unit operation, which includes one or more post-capture chromatography systems; and (d) The fourth unit operation. The fourth unit operation includes an ultrafiltration system and a diafiltration system. The system according to claim 24, further comprising a fifth unit operation located between the first unit operation and the second unit operation, wherein the fifth unit operation includes a subroutine for performing virus inactivation system. The system according to claim 25, wherein the subsystem is a tubular flow reactor. The system according to claim 24, further comprising a sixth unit operation located between the third unit operation and the fourth unit operation, wherein the sixth unit operation includes a second unit operation for performing virus filtering. Subsystem. The system according to claim 24, wherein the one or more capture chromatography systems and the one or more post-capture chromatography systems include chromatography media, and the chromatography media includes resin beads, porous membranes, or Nano fiber. The system according to claim 28, wherein the chromatography medium is functionalized for affinity chromatography, cation exchange chromatography, anion exchange chromatography, hydrophobic interaction chromatography, or mixed mode chromatography. The system according to claim 29, wherein the affinity chromatography medium is a protein A-based resin. The system of claim 24, wherein the one or more capture chromatography columns and the one or more post-capture chromatography systems are periodic countercurrent chromatography systems (PCCS). The system of claim 26, wherein the tubular flow reactor has a defined minimum residence time of at least 30 minutes. The system according to claim 27, wherein the second subsystem for performing virus filtration is a pressurized container, and the pressurized container includes a virus removal filter and a sterilization filter. The system according to claim 33, wherein the pressurized container is a single-use, closed and sterilizable container. The system according to claim 33, wherein the virus removal filter is an ultra filter or a nano filter. The system according to claim 24, wherein the first unit operation includes an outlet connected to the inlet of the second unit operation, and wherein the second unit operation includes an outlet connected to the inlet of the third unit operation , Wherein the third unit operation includes an outlet connected to the inlet of the fourth unit operation. The system according to claim 24, further comprising a buffer between each of the first unit operation, the second unit operation, the third unit operation, and the fourth unit operation . The system according to claim 24, wherein the recombinant protein is an antibody or an antigen-binding fragment thereof. The system according to claim 24, wherein the system is on a sliding rail. The system according to claim 24, wherein the system is airtight. The system according to claim 24, further comprising a seventh unit operation, the seventh unit operation comprising a third sub-system, wherein the third sub-system includes an online excipient for formulating a therapeutic drug substance.

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